Survivability of a probiotic Lactobacillus casei in the gastrointestinal tract of healthy human volunteers and its impact on the faecal microflora

Aim: The aim of this study was to measure the gastrointestinal survival of Lactobacillus casei and its impact on the gut microflora in healthy human volunteers. Methods and Results: Twenty healthy volunteers took part in a double-blind placebo-controlled probiotic feeding study (10 fed probiotic, 10 fed placebo). The probiotic was delivered in two 65 ml aliquots of fermented milk drink (FMD) daily for 21 days at a dose of 8.6 +/- 0.1 Log(10)Lact. casei CFU ml(-1) FMD. Faecal samples were collected before, during and after FMD or placebo consumption, and important groups of faecal bacteria enumerated by fluorescent in situ hybridization (FISH) using oligonucleotide probes targeting the 16S rRNA. The fed Lact. casei was enumerated using selective nutrient agar and colony identity confirmed by pulsed field gel electrophoresis. Seven days after ingestion of FMD, the Lact. casei was recovered from faecal samples taken from the active treatment group at 7.1 +/- 0.4 Log(10) CFU g(-1) faeces (mean +/- SD, n = 9) and numbers were maintained at this level until day 21. Lact. casei persisted in six volunteers until day 28 at 5.0 +/- 0.9 Log(10) CFU g(-1) faeces (mean +/- SD, n = 6). Numbers of faecal lactobacilli increased significantly upon FMD ingestion. In addition, the numbers of bifidobacteria were higher on days 7 and 21 than on days 0 and 28 in both FMD fed and placebo fed groups. Consumption of Lact. casei had little discernible effect on other bacterial groups enumerated. Conclusions: Daily consumption of FMD enabled a probiotic Lact. casei strain to be maintained in the gastrointestinal tract of volunteers at a stable relatively high population level during the probiotic feeding period. Significance and Impact of the Study: The study has confirmed that this probiotic version of Lact. casei survives well within the human gastrointestinal tract.

In vitro examination of the effect of Orlistat on the ability of the faecal microbiota to utilize dietary lipids

Orlistat is an anti-obesity treatment with which several gastrointestinal (GI) side-effects are commonly associated in the initial stages of therapy. There is no physiological explanation as to why two-thirds of those who take the drug experience one or more side-effects. It has been hypothesized that the GI microbiota may protect from or contribute to these GI disturbances. Using in vitro batch culture and human gut model systems, studies were conducted to determine whether increased availability of dietary lipids and/or orlistat affect the composition and/or activity of the faecal microbiota. Results from 24-h batch culture fermentation experiments demonstrated no effect of orlistat in the presence or absence of a dietary lipid (olive oil) on the composition of bacterial communities [as determined by fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses], but did show there was great variability in the lipolytic activities of the microbiotas of individuals, as determined by gas chromatography analysis of long-chain fatty acids in samples. Subsequent studies focused on the effect of orlistat in the presence and absence of lipid in in vitro human gut model systems. Systems were run for 14 days with gut model medium (GMM) only (to steady state, SS), then fed at 12-h intervals with 50 mg orlistat, 2 g olive oil or a mixture of both for 14 days. FISH and DGGE were used to monitor changes in bacterial populations. Bacteria were cultivated from the GMM only (control) systems at SS. All strains isolated were screened for lipolytic activity using tributyrin agar. FISH and DGGE demonstrated that none of the compounds (singly or in combination) added to the systems had any notable effect on microbial population dynamics for any of the donors, although Subdoligranulum populations appeared to be inhibited by orlistat in the presence or absence of lipid. Orlistat had little or no effect on the metabolism of indigenous and added lipids in the fermentation systems, but there was great variability in the way the faecal microbiotas of the donors were able to degrade added lipids. Variability in lipid degradation could be correlated with the number and activity of isolated lipolytic bacteria. The mechanism by which orlistat and the GI microbiota cause side-effects in individuals is unknown, but several hypotheses have been proposed to account for their manifestation. The demonstration of great variability in the lipolytic activity of microbiotas to degrade lipids led to a large-scale cultivation-based study of lipolytic/lipase-positive bacteria present in the human faecal microbiota. Of 4,000 colonies isolated from 15 donors using five different agars, 378 strains were identified that had lipase activity. Molecular identification of strains isolated from five donors demonstrated that lipase activity is more prevalent in the human GI microbiota than previously thought, with members of the phyla Firmicutes, Bacteroidetes and Actinobacteria identified. Molecular identification and characterization of the substrate specificities of the strains will be carried out as part of ongoing work.

Quantification of major camel milk proteins by capillary electrophoresis

Proteins from dromedary camel milk (CM) produced in Europe were separated and quantified by capillary electrophoresis (CE). CE analysis showed that camel milk lacks b-lactoglobulin and consists of high concentration of a-lactalbumin (2.01 ± 0.02 mg mL-1), lactoferrin (1.74 ± 0.06 mg mL-1) and serum albumin (0.46 ± 0.01 mg mL-1 ). Among caseins, the concentration of b-casein (12.78 ± 0.92 mg mL-1) was found the highest followed by a-casein (2.89 ± 0.29 mg mL-1) while k-casein represented only minor amount (1.67 ± 0.01 mg mL-1). These results were in agreement with sodium dodecyl sulphatepolyacrylamide gel electrophoresis patterns. Overall, CE offers a quick and reliable method for the determination of major CM proteins, which may be responsible for the many nutritional and health properties of CM.

Proteinograma sérico de cães sadios e com linfoma obtido por eletroforese em gel de poliacrilamida (SDS-PAGE)

Foram avaliadas amostras de soro sanguíneo de 10 cães sadios e de 12 com linfoma, utilizando-se a eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio. Houve diferença entre as médias dos teores de proteína total de cães sadios, 7,68g/dL±0,46 e de cães com linfoma, 7,93g/dL±2,49. As concentrações de IgA e IgG não foram diferentes entre os grupos. Os teores das proteínas de pesos moleculares 142000, 110000, 52000, 49000, 24000 e 18000 dáltons foram mais elevados em cães com linfoma. Os cães com linfoma apresentaram concentrações mais elevadas de ceruloplasmina, 43,95mg/dL±18,19, e haptoglobina, 554mg/dL±449,51, e menores de albumina, 2908mg/dL±476,67, em comparação aos cães sadios (ceruloplasmina: 3,42mg/dL±7,44; haptoglobina: 94,54mg/dL±59,50 e albumina: 4207mg/dL±206,18). Conclui-se que concentrações séricas mais elevadas de ceruloplasmina e haptoglobina e menores de albumina podem estar associadas ao linfoma em cães.

Influência do puerpério sobre o proteinograma sérico de caprinos da raça Saanen obtido por eletroforese em gel de poliacrilamida

Aiming to evaluate the puerperal influence on the proteinogram of Saanen goats, 108 samples of blood serum from 12 goats were collected, and the results were presented at nine times: just after parturition, 1, 3, 5, 7, 10, 15, 21 and 30 days after parturition. Total amount of serum proteins were determined by the biuret technique, and the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to the protein fractionation. In this last method, 17 protein bands were observed, from which molecular weights varied between 25 KDa and 275 KDa. In addition, it was possible to identify the following protein fractions: immunoglobulin A (180 KDa), ceruloplasmin (115 KDa), transferrin (79 KDa), albumin (65 KDa), heavy-chain immunoglobulin G (58 KDa), haptoglobin (45 KDa), acid glycoprotein (37 KDa) and light-chain immunoglobulin G (28 KDa). Another 9 nonidentified protein fractions presented, each molecular weights equal to 275 KDa, 140 KDa, 125 KDa, 103 KDa, 95 KDa, 41 KDa, 35 KDa, 30 Kda and 25 KDa. The results allow us to conclude that by the first week of puerperium, an improvement of acid glycoprotein occurs, whereas those others protein fractions do not suffer any puerperal influence.

Genetic polymorphism of Streptococcus mutans in Brazilian family members

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Imunodifusão em gel de ágar com polissacarídeos de membrana de Brucella abortus 1119-3 no diagnóstico da brucelose bovina

Comparou-se a prova de imunodifusão em gel de ágar (IDGA), utilizando extrato polissacarídico (POLI O), obtido da amostra de B. abortus 1119-3, com os testes de soroaglutinação rápida em placa, de soroaglutinação lenta em tubos, de antígeno acidificado e de 2-mercaptoetanol para o diagnóstico da brucelose bovina. O IDGA mostrou alta especificidade, porém sensibilidade inferior aos métodos convencionais.

An improved electrophoretic method for a screening program for haemoglobinopathies

A method for a screening program for haemoglobinopathies in a starch agar gel mixed with saponin is presented. Normal and abnormal blood containing haemoglobins S, C, I, M Boston, D Punjab, beta thalassaemia major and beta thalassaemia minor, were applied, in a tray with the capacity for 100 samples. The electrophoresis was performed in 45 min using 300 V. This method offers special advantages for the examination of a large number of samples, using a small amount of whole blood and without the previous preparation of haemoglobin solution.

Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiine cichlid fish, Oreochromis niloticus

In this study, we report the cloning and nucleotide sequence of PCR-generated 5S rDNA from the Tilapiine cichlid fish, Oreochromis niloticus. Two types of 5S rDNA were detected that differed by insertions and/or deletions and base substitutions within the non-transcribed spacer (NTS). Two 5S rDNA loci were observed by fluorescent in situ hybridization (FISH) in metaphase spreads of tilapia chromosomes. FISH using an 18S rDNA probe and silver nitrate sequential staining of 5S-FISH slides showed three 18S rDNA loci that are not syntenic to the 5S rDNA loci.

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

Antimicrobial resistance and persistence of Staphylococcus epidermidis clones in a Brazilian university hospital

Oxacillin is an alternative for the treatment of Staphylococcus spp. infections; however, resistance to this drug has become a major problem over recent decades. The main objective of this study was to epidemiologically characterize coagulase-negative staphylococci (CoNS) strains recovered from blood of patients hospitalized in a Brazilian teaching hospital. Oxacillin resistance was analyzed in 160 strains isolated from blood culture samples by phenotypic methods, detection of the mecA gene, and determination of intermediate sensitivity to vancomycin on brain heart infusion agar supplemented with 4 and 6 μg/mL vancomycin. In addition, characterization of the epidemiological profile by staphylococcal cassette chromosome mec (SCC. mec) typing and clonal analysis by pulsed-field gel electrophoresis (PFGE) were performed. The mecA gene was detected in 72.5% of the isolates. Methicillin-resistant CoNS isolates exhibited the highest minimum inhibitory concentrations and multiresistance when compared to methicillin-susceptible CoNS strains. Typing classified 32.8% of the isolates as SCC. mec I and 50% as SCC. mec III. PFGE typing of the SCC. mec III Staphylococcus epidermidis isolates identified 6 clones disseminated in different wards that persisted from 2002 to 2009. The high oxacillin resistance rates found in this study and clonal dissemination in different wards highlight the importance of good practices in nosocomial infection control and of the rational use of antibiotic therapy in order to prevent the dissemination of these clones. © 2013 Elsevier Inc.

Características fenotípicas e genotípicas de Escherichia coli isoladas de queijos produzidos a partir de leite não pasteurizado

Pós-graduação em Medicina Veterinária - FCAV

Inquérito sorológico de brucelose ovina em cabanhas registradas da Região Centro-Oeste do Estado de São Paulo, através das técnicas de imunodifusão em gel de ágar e ELISA

Pós-graduação em Medicina Veterinária - FMVZ

Selênio em tilápia do Nilo utilizando eletroforese em gel e espectrometria atômica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hemoglobin patterns in different populations of Synbranchus marmoratus Bloch, 1795 (Pisces, Synbranchidae)

1. 1. Total hemolysates of Synbranchus marmoratus Bloch, 1795 captured at four different sites in the State of São Paulo, Brazil, showed two different hemoglobin phenotypes when submitted to agar-starch gel electrophoresis on glass slides in basic buffer. 2. 2. Phenotype I was characterized by 3 hemoglobin bands. When the total hemolysate was submitted to cellulose acetate electrophoresis in basic buffer containing 6 M urea and β-mercaptoethanol, Phenotype I showed four globins of the α 1, α 2, β and γ types, with 11.9 ± 1.9 g% total hemoglobin, 45.3 ± 3.6% globular volume, and 26.8 ± 4.4% mean corpuscular hemoglobin concentration (MCHC). 3. 3. Phenotype II showed three groups of hemoglobins, with a total of up to 12 hemoglobin bands. When the total hemolysate was submitted to cellulose acetate electrophoresis in basic buffer containing 6 M urea and β-mercaptoethanol, phenotype II showed five types of globins, denoted types α 1, α 2, γ 1, γ 2 and β, having electrophoretic positions different from those of Phenotype I globins, with 18.1 ± 3.3% total hemoglobin, 47.9 ± 6.4% globular volume, and 37.8 ± 4.4% MCHC. 4. 4. The distribution of the specimens having the two hemoglobin phenotypes is associated with the different geomorphological provinces of the State of São Paulo, suggesting the existence of at least two populational groups of Synbranchus marmoratus. © 1986.